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Detection of cucurbit viruses in Oklahoma combining EDNA with Multiplex RT-PCR coupled with High Resolution Melting
Lizbeth Pena Zuniga: Oklahoma State University; Andres Espindola: Oklahoma State University; Hassan Melouk: USDA ARS; Akhtar Ali: Univ of Tulsa; Francisco Ochoa Corona: Oklahoma State University; Kitty Cardwell: Oklahoma State University
<div>Cultivated acreage of cucurbits in Oklahoma has decreased mainly due to viral diseases. This research combines Electronic probe Diagnostic Nucleic acid Analysis (EDNA) for detection of 17 reported viruses infecting cucurbits with Multiplex RT-PCR and High Resolution Melting (HRM). EDNA<b> </b>is a next generation sequencing (NGS) tool that generates specific electronic probes (e-probes). A mock positive controls database is created <i>in silico</i> with MetaSim using pathogen sequences retrieved from GenBank to mimic single and multiple infections. To avoid false positives, e-probes are made more specific by BLAST alignments of sequences from published databases of near neighbors, to eliminate homologous sequences. Once a sample NGS output is retrieved, the probes and control sequences are used to query the sequence for matches, indicating positive detection. Next, the sample is tested <i>in vitro</i> by a Multiplex RT-PCR+HRM for confirmation and discrimination based on PCR products melting profiles. EDNA probes and multiplex RT-PCR+HRM of nine out of 17cucurbit infecting viruses are presented: <i>Cucurbit green mottle mosaic virus</i> (CGMMV), <i>Cucumber mosaic virus </i>(CMV I and CMV II), <i>Cucurbit aphid-borne yellow virus </i>(CABYV), <i>Cucurbit yellow stunting disorder virus</i> (CYSDV), <i>Melon necrotic spot virus</i> (MNSV), <i>Papaya ringspot virus</i> (PRSV), <i>Squash mosaic virus</i> (SqMV), <i>Watermelon mosaic virus</i> (WMV), and <i>Zucchini yellow mosaic virus </i>(ZYMV). EDNA-Cucurbits can be combined with Multiplex RT-PCR+HRM for virus monitoring in microbial forensics and biosecurity, and for routine diagnostics and epidemiological studies of cucurbit crops.</div>

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